3.1 Rational design of FGF2 variants
Selection of an appropriate modification site is important for the retention of biological activity of PEGylated proteins. The surface of FGF2 contains two native cysteines, Cys69 and Cys87, which are surface exposed and spatially distal from both the receptor and heparin binding regions. We separately mutated each cysteine to alanine, thus generating two FGF2 containing only a single surface-exposed cysteine (compounds1 and 2 ). And two surface-exposed residues, Lys129 and Phe17, near the heparin- and receptor-binding regions, respectively, were also changed to cysteines (compounds 3 and 4 ) (Figure 1A ). The resulting FGF2 variants, each containing a single cysteine for PEGylation, were engineered and tested: FGF2C69A (compound 1 ), FGF2C87A (compound 2 ), FGF2C69A/C87A/F17C (compound 3 ), and FGF2C69A/C87A/K129C (compound 4 ) (Figure 1 B and E ).
The activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) is a key point of convergence for many signaling pathways, and the biological response of extracellular stimuli including FGF2 requires ERK1/2 activation (Johnson et al. , 2002). Hence, signaling of the ERK1⁄2 pathway was evaluated by immunoblotting to assay the activity of FGF2 and its mutants. The cellular phosphorylated ERK1⁄2 (p-ERK1/2) levels were increased with native FGF2 compared with the control group, and the same increase occurred with compounds 1 and 2(Figure 1 C-D ). A slight reduction in the cellular level of p-ERK1⁄2 was observed for compounds3 and 4 (Figure 1 E-F ). These results indicated that both the receptor- and heparin-binding regions are indeed important for FGF2 activity and that single site mutation distal from the binding domains has little effect on protein activity.